In both animals and plants, the formation of a polar axis is one of the first steps during embryogenesis. Cell fate must then be specified and maintained along this axis in order for proper development to occur. In animals, inherited and somatic mutations in the genes that regulate this process can lead to many types of cancers. Therefore it is important to identify and understand the mechanisms by which these proteins act. We have identified a mutation in the Arabidopsis TOPLESS gene (tpl-1) that cause the transformation of the apical half of the embryo (the shoot) into a second basal half (the root). The apical half of early tpl- 1 embryos express shoot fate genes, but these are lost later in embryogenesis and replaced by the expression of root fate genes. The TPL protein shows structural similarity to known co-repressors, and tpl-1 can be suppressed by mutations in the putative co-activator HAG1, a GCN5-1ike protein. This has lead us to a hypothesis wherein TPL acts as a transcriptional co-repressor, whose role is to fix the identity of the shoot by repressing genes required for root formation in the apical half of the embryo. We propose to test this hypothesis and use TPL and HAG to identify other key regulators of embryonic polarity. In AIM 1, we will test if TPL can act as a transcriptional repressor using both yeast and in planta transcriptional repression assays and determine if the tpl-1 mutation affects the ability of TPL to repress transcription. We will also fuse TPL to known activation and repression domains and analyze the phenotypic consequences in transgenic Arabidopsis. In AIM2, we will use microarray analysis to compare wild-type, tpl-1 and tpl-1;hag1 mutant embryos to identify the common downstream genes of TPL and HAG1. We will use inducible forms of TPL, tpl-1 and HAG1 to place these downstream genes into a temporal context. In AIM3 we will perform an enhancer, a suppressor, and a modular misexpression screen on tpl-1 to identify other genes in the TPL/HAG 1 pathway. These experiments will shed light on one of the mechanisms by which plants specify and maintain cell types during embryogenesis. It will allow us to compare the proteins and logic used in plants to those used in cognate processes in animal development, leading to a better understanding of both.